It is known that xanthine oxidase (EC 1.2.3.2) catalyzes the oxidation of the substrate thereof such as hypoxanthine to form uric acid and hydrogen peroxide and that the enzyme exists in milk. The enzyme derived from milk is available.
The determination of the substrate for an oxidase by oxidizing the substrate by the action of the oxidase and determining the amount of reaction product stoichiometrically formed is generally conducted. For example, it has been known that hypoxanthine is oxidized by the action of xanthine oxidase to form uric acid and hydrogen peroxide. Therefore, it is easily understood that the determination of the substrate can be performed by determining the amount of formed uric acid or hydrogen peroxide.
When the substrate is determined from the amount of a product obtained by oxidizing the substrate by the action of oxidase, the determination is generally carried out by reacting the formed hydrogen peroxide with a coloring reagent and determining the amount of the formed hydrogen peroxide by measuring the absorbancy of the reaction solution in visible ray region.
When the substrate is oxidized by the action of xanthine oxidase derived from milk and the formed hydrogen peroxide is introduced into a pigment system, the formed pigment is unstable and is decomposed in a brief period of time. Therefore, the substrate cannot be determined by the change in absorbancy in visible ray region. Accordingly, the substrate is generally determined by the change in absorbancy in ultraviolet region based on the formation of uric acid without the determination of hydrogen peroxide.
After studying the process for producing a xantine oxidase by fermentation, the present inventors have found that xanthine oxidase is produced by culturing a microorganism belonging to the genus Enterobacter and capable of producing xanthine oxidase in a nutrient medium.
It has been found that when the substrate is oxidized by the action of xanthine oxidase derived from microorganism and the formed hydrogen peroxide is introduced into a pigment system, the formed pigment is stable and the substrate in a sample can be determined by measuring the absorbancy in visible ray region based on the pigment.
It has been known that the enzyme derived from microorganism belonging to the genus Pseudomonas, Escherichia, Arthrobacter, Nocardia or the like exhibits a weak activity of xanthine oxidase. [J. Bacteriology, 130, 1175 (1977)]. However, these enzymes do not have a sufficiently strong activity for the determination of the substrate.